DESCRIPTION: Alcoholic liver disease (ALD) is the second highest cause of liver transplants in the United States, yet the mechanisms that underlie ethanol-induced liver damage remain poorly understood. Clinical studies of ALD recognize intestinal dysfunction as a critical player in the progression of disease. Chronic ethanol consumption induces transient losses in the intestinal barrier, which correlate with significant increases in circulating microbial byproducts such as lipopolysaccharide (LPS). Increases in LPS are associated with activation of toll like receptor 4 (TLR4), which stimulates the release of inflammatory cytokines from intestinal and liver resident macrophages, and results in the dysfunction of the liver parenchyma. Cobalt protoporphyrin (CoPP), a global inducer of heme oxygenase-1 (HO-1), inhibits the production of inflammatory cytokines in Kupffer cells and significantly reduces markers of liver injury. However, CoPP also induces HO-1 in the hepatocytes and the intestinal epithelium. Preliminary data in caco-2 cells suggest that epithelial HO-1 expression protects tight junctions. We hypothesize that the induction of HO-1 within the intestinal epithelium protects the liver from inflammation and damage by preserving the function of the intestinal tight junctions during ethanol feeding. To investigate the role of HO-1 in maintaining the intestinal barrier, the localization of tight junctions will be determined in the jejunum, ileum, proximal, and distal colo. The modified lactulose-mannitol test will be used to assess permeability of the fore, mid, and hindgut. Barrier measurements will be compared with circulating endotoxin to determine the impact of global HO-1 induction on endotoxin translocation. To determine the impact of epithelial HO-1 on liver protection, conditional HO-1 knockout mice will be generated for the intestinal epithelium, and the HO-1 will be induced with CoPP during the chronic ethanol diet. Hepatocyte, and monocyte conditional HO-1 knockouts will be generated to determine the impact of HO-1 in the liver parenchyma. The permeability of caco-2 monolayers in response to ethanol will be determined under conditions with or without CoPP and compared with the localization and relative expression of tight junction proteins. The impact of HO-1 on tight junctions will be determined by treating with inhibitors of HO-1 expression or enzymatic activity. HO-1 catalyzes the degradation of heme into carbon monoxide, biliverdin, and molecular iron, which is associated with upregulation of ferritin heavy chain. The impact of these products on the localization of tight junctions and barrier permeability will be determined using pharmacologic inhibitors, overexpression plasmids, shRNA and scrambled controls. During these experiments, the transepithelial electrical resistance, immunohistochemistry and FITC-dextran permeability assays will be used to assess barrier function.